Himmat Grewal

Androgens and stroke: good, bad or indifferent?

Cerebral ischemia caused by loss of blood supply to the brain during cardiac arrest or stroke are major causes of death and disability. Biological sex is an important factor in predicting vulnerability of the brain to an ischemic insult, with males being at higher risk for cardio-cerebrovascular events than females of the same age. However, relative incidence of stroke between the genders appears to normalize at advanced ages. Therefore, many scientists have focused on the mechanisms of sex differences in outcome following brain ischemic injury, with a particular emphasis on the role of sex steroids. The majority of studies indicate that female sex steroids, such as estrogen and progesterone, play important roles in the relative neuroprotection following cerebral ischemia observed in females. However, less is known about male sex steroids and brain damage. This review describes the state of our knowledge of androgen-related contributions to neurological injury and recovery following cerebral ischemia that occurs following stroke. Experimental studies examining the effects of castration, androgenic agonists and antagonists and aging provide valuable insights into the role of androgens in clinical outcome following cerebrovascular events.

Alterations in Purkinje cell GABAA receptor pharmacology following oxygen and glucose deprivation and cerebral ischemia reveal novel contribution of β1‐subunit‐containing receptors

Cerebellar Purkinje cells (PCs) are particularly sensitive to cerebral ischemia, and decreased GABAA receptor function following injury is thought to contribute to PC sensitivity to ischemia‐induced excitotoxicity. Here we examined the functional properties of the GABAA receptors that are spared following ischemia in cultured Purkinje cells from rat and in vivo ischemia in mouse. Using subunit‐specific positive modulators of GABAA receptors, we observed that oxygen and glucose deprivation (OGD) and cardiac arrest‐induced cerebral ischemia cause a decrease in sensitivity to the β2/3‐subunit‐preferring compound, etomidate. However, sensitivity to propofol, a β‐subunit‐acting compound that modulates β1–3‐subunits, was not affected by OGD. The α/γ‐subunit‐acting compounds, diazepam and zolpidem, were also unaffected by OGD. We performed single‐cell reverse transcription–polymerase chain reaction on isolated PCs from acutely dissociated cerebellar tissue and observed that PCs expressed the β1‐subunit, contrary to previous reports examining GABAA receptor subunit expression in PCs. GABAA receptor β1‐subunit protein was also detected in cultured PCs by western blot and by immunohistochemistry in the adult mouse cerebellum and levels remained unaffected by ischemia. High concentrations of loreclezole (30 μm) inhibited PC GABA‐mediated currents, as previously demonstrated with β1‐subunit‐containing GABAA receptors expressed in heterologous systems. From our data we conclude that PCs express the β1‐subunit and that there is a greater contribution of β1‐subunit‐containing GABAA receptors following OGD.

Region-specific role for GluN2B-containing NMDA receptors in injury to Purkinje cells and CA1 neurons following global cerebral ischemia

Motor deficits are present in cardiac arrest survivors and injury to cerebellar Purkinje cells (PCs) likely contribute to impairments in motor coordination and post-hypoxic myoclonus. N-Methyl-D-aspartic acid (NMDA) receptor-mediated excitotoxicity is a well-established mechanism of cell death in several brain regions, but the role of NMDA receptors in PC injury remains understudied. Emerging data in cortical and hippocampal neurons indicate that the GluN2A-containing NMDA receptors signal to improve cell survival and GluN2B-containing receptors contribute to neuronal injury. This study compared neuronal injury in the hippocampal CA1 region to that in PCs and investigated the role of NMDA receptors in PC injury in our mouse model of cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Analysis of cell density demonstrated a 24% loss of PCs within 24 h after 8 min CA/CPR and injury stabilized to 33% by 7 days. The subunit promiscuous NMDA receptor antagonist MK-801 protected both CA1 neurons and PCs from ischemic injury following CA/CPR, demonstrating a role for NMDA receptor activation in injury to both brain regions. In contrast, the GluN2B antagonist, Co 101244, had no effect on PC loss while protecting against injury in the CA1 region. These data indicate that ischemic injury to cerebellar PCs progresses via different cell death mechanisms compared to hippocampal CA1 neurons.

Sex Steroids Do Not Modulate TRPM2-Mediated Injury in Females following Middle Cerebral Artery Occlusion

TRPM2 is an ion channel that is activated by ischemia in stroke and contributes to neuronal injury only in males. We tested whether the lack of TRPM2 activation following stroke in females is caused by differences in sex steroids. Abstract Calcium-permeable transient receptor potential M2 (TRPM2) ion channel activation contributes to cerebral ischemic injury specifically in males. In male mice, circulating androgens are required for TRPM2 inhibition with clotrimazole (CTZ) to provide protection following experimental stroke. Sufficient levels of circulating androgens are necessary to support ischemia-induced activation of poly ADP ribose polymerase (PARP) and consequent activation of TRPM2 channels. In this study, we tested whether differences in sex steroids contribute to the lack of CTZ neuroprotection in females. Middle cerebral artery occlusion (MCAO) was performed using adult female mice that were hormonally intact, ovariectomized (OVX) or dihydrotestosterone (DHT) treated. CTZ or vehicle was administered at the time of reperfusion, animals were euthanized 24 h later and brains and serum were collected. Infarct analysis revealed no effect of CTZ in intact females or females lacking endogenous sex steroids (OVX). Interestingly, treatment of female mice with the potent androgen receptor agonist DHT had no effect on ischemic injury and did not permit CTZ neuroprotection. Similarly, DHT-treated females did not exhibit increased levels of ADPribose, the TRPM2 ligand generated by PARP, following ischemia. No differences in TRPM2 or androgen receptor expression were observed between males and females. These data suggest that the lack of TRPM2 activation in females following experimental stroke is not due to the presence of estrogen or the absence of androgens. In conclusion, our data demonstrate that while circulating androgens are necessary for PARP-mediated TRPM2 injury in males, they are not sufficient to produce TRPM2 activation in females.

Juvenile striatal white matter is resistant to ischemia‐induced damage

White matter injury following ischemic stroke is a major cause of functional disability. Injury to both myelinated axons and oligodendrocytes, the myelin producing cells in the central nervous system, occurs in experimental models of ischemic stroke. Age‐related changes in white matter vulnerability to ischemia have been extensively studied and suggest that both the perinatal and the aged periods are times of increased white matter vulnerability. However, sensitivity of white matter following stroke in the juvenile brain has not been evaluated. Interestingly, the late pediatric period is an important developmental stage, as it is the time of maximal myelination. The current study demonstrates that neurons in late pediatric/juvenile striatum are vulnerable to ischemic damage, with neuronal injury being comparable in juvenile and adult mice following ischemia. By contrast, actively myelinating striatal oligodendrocytes in the juvenile brain are resistant to ischemia, whereas adult oligodendrocytes are quite sensitive. As a result, myelin sheaths are remarkably intact and axons survive well in the injured striatum of juvenile mice. In addition to relative resistance of juvenile white matter, other glial responses were very different in juvenile and adult mice following cerebral ischemia, including differences in astrogliosis, fibrosis, NG2‐cell reactivity, and vascular integrity. Together, these responses lead to long‐term preservation of brain parenchyma in juvenile mice, compared to severe tissue loss and scarring in adult mice. Overall, the current study suggests that equivalent ischemic insults may result in less functional deficit in children compared to adults and an environment more conducive to long‐term recovery. GLIA 2016;64:1972–1986

Col1a1+ perivascular cells in the brain are a source of retinoic acid following stroke

BackgroundPerivascular stromal cells (PSCs) are a recently identified cell type that comprises a small percentage of the platelet derived growth factor receptor-β+ cells within the CNS perivascular space. PSCs are activated following injury to the brain or spinal cord, expand in number and contribute to fibrotic scar formation within the injury site. Beyond fibrosis, their high density in the lesion core makes them a potential significant source of signals that act on neural cells adjacent to the lesion site.ResultsOur developmental analysis of PSCs, defined by expression of Collagen1a1 in the maturing brain, revealed that PSCs first appear postnatally and may originate from the meninges. PSCs express many of the same markers as meningeal fibroblasts, including expression of the retinoic acid (RA) synthesis proteins Raldh1 and Raldh2. Using a focal brain ischemia injury model to induce PSC activation and expansion, we show a substantial increase in Raldh1+/Raldh2+ PSCs and Raldh1+ activated macrophages in the lesion core. We find that RA levels are significantly elevated in the ischemic hemisphere and induce signaling in astrocytes and neurons in the peri-infarct region.ConclusionsThis study highlights a dual role for activated, non-neural cells where PSCs deposit fibrotic ECM proteins and, along with macrophages, act as a potentially important source of RA, a potent signaling molecule that could influence recovery events in a neuroprotective fashion following brain injury.

Delayed inhibition of tonic inhibition enhances functional recovery following experimental ischemic stroke

The current study focuses on the ability to improve cognitive function after stroke with interventions administered at delayed/chronic time points. In light of recent studies demonstrating delayed GABA antagonists improve motor function, we utilized electrophysiology, biochemistry and neurobehavioral methods to investigate the role of α5 GABAA receptors on hippocampal plasticity and functional recovery following ischemic stroke. Male C57Bl/6 mice were exposed to 45 min transient middle cerebral artery occlusion and analysis of synaptic and functional deficits performed 7 or 30 days after recovery. Our findings indicate that hippocampal long-term potentiation (LTP) is impaired 7 days after stroke and remain impaired for at least 30 days. We demonstrate that ex vivo administration of L655,708 reversed ischemia-induced plasticity deficits and importantly, in vivo administration at delayed time-points reversed stroke-induced memory deficits. Western blot analysis of hippocampal tissue reveals proteins responsible for GABA synthesis are upregulated (GAD65/67 and MAOB), increasing GABA in hippocampal interneurons 30 days after stroke. Thus, our data indicate that both synaptic plasticity and memory impairments observed after stroke are caused by excessive tonic GABA activity, making inhibition of specific GABA activity at delayed timepoints a potential therapeutic approach to improve functional recovery and reverse cognitive impairments after stroke.